Forms of prostate specific antigens and methods for their detection

ABSTRACT

Inactive precursor forms of PSA (pPSA) have been identified to exist in serum and tissues of patients with prostate cancer. Antibodies specific for pPSA are provided. Methods for detecting inactive precursors of PSA in human physiological fluid and tissues are also provided, as well as diagnostic kits and methods useful in the diagnosis and management of prostate cancer.

[0001] This is a continuation-in-part of application Ser. No.09/302,965, filed on Apr. 30, 1999, which in turn is acontinuation-in-part of application Ser. No. 09/251,686, filed on Feb.17, 1999, which in turn is a continuation of application Ser. No.08/846,408, filed on Apr. 30, 1997. The content of the application Ser.Nos. 09/251,686, 09/302,965, and 08/846,408 are incorporated herein intheir entirety by reference.

FIELD OF THE INVENTION

[0002] The present invention relates generally to the detection andidentification of proteins, as well as various forms and subunits ofproteins, which have the potential utility as diagnostic markers. Inparticular, the present invention relates to the detection of inactiveprecursor forms of prostate specific antigens, and antibodies that arecapable of preferentially binding to precursor forms of prostatespecific antigens.

BACKGROUND OF THE INVENTION

[0003] The measurement of serum prostate specific antigen (PSA) iswidely used for the screening and early detection of prostate cancer¹⁻³.Serum PSA that is measurable by current clinical immunoassays existsprimarily as either the free “non-complexed” form (free PSA), or as acomplex with α₁-antichymotrypsin (ACT)^(4,5). The ratio of free to totalPSA in serum has been demonstrated to significantly improve thediscrimination of PCa from benign prostatic diseases, with higher ratioscorrelating with a lower risk of prostate cancer^(6,7). While benignprostate hyperplasia (BPH) is the most common benign prostatic diseaseit is recognized that other benign diseases such as prostatitis,prostatic infarct and prostatic injury can also elevate serum PSA andcause changes in the free to total PSA ratio.

[0004] The biological mechanism for the variable levels of free PSA inserum is unknown. The serum PSA that has become complexed is likely tobe relatively homogeneous since this represents enzymatically active,intact PSA. The PSA released from the PSA-ACT complex in prostate cancer(Pca) and benign prostate hyperplasia (BPH) serum was found to beindistinguishable from seminal plasma PSA, which confirms thisassumptions⁸. It follows that free PSA may offer better biochemicalinsight, and that a characterization of the molecular forms of free PSAcould help elucidate their prostatic origin and mechanism of releaseinto the serum. However, attempts to purify and characterize the lowlevels of PSA from serum in the diagnostically relevant range near 10ng/ml have not generally been considered feasible with currenttechnologies. So far, studies have focused primarily on serum from menwith unusually high levels of PSA, with 100's or 1000's of ng/ml PSA.However, other groups have failed to identify pPSA in serum containingthese high levels of PSA^(9,10). While studies using high serum PSAlevels are suggestive, they suffer a common drawback in that this PSAmay not reflect the kind or percentage of PSA that is typically presentin the early stages of disease, where PSA is 10 ng/ml or less. PSAreleased from large primary tumor lesions or metastatic disease may havedifferent biochemical properties than PSA released from early, possiblylower grade disease. Therefore, in order to be useful for clinicaldetection of early prostate cancer, the truncated pPSA forms would haveto be present at significant levels in serum with diagnosticallyrelevant levels of total PSA near 10 ng/ml.

[0005] Accordingly, a need exists to characterize different forms offree PSA present in prostate cancer serum with diagnostically relevantlevels of total PSA near 10 ng/ml. A need also exists to determine thediagnostic potential of these pPSA forms in prostate cancer detection.In addition, since any characterization of the free PSA forms in serummust necessarily depend on the development of mAbs, a need also existsfor developing antibodies that are specific for these pPSA forms.

SUMMARY OF THE INVENTION

[0006] The present invention is based on the successful expression ofchimeric pPSA protein in mammalian cells. It is herein demonstrated forthe first time that PSA is secreted into the spent media by mammaliancells as proPSA. The proPSA thus secreted is enzymatically inactive andstable in the media. Therefore, vectors of the present invention may beused to generate proPSA polypeptides.

[0007] Accordingly, one aspect of the present invention provides achimeric expression vector comprising a nucleic acid molecule. Thenucleic acid molecule encodes a pPSA polypeptide. The nucleic acidmolecule is preferably operably linked to control sequences which arerecognized by a host cell that is transformed with the expressionvector. The host cell is preferably derived from a mammalian source.

[0008] ProPSA polypeptides, as well as variants and subunits thereof,produced by the methods of the present invention can be used to producepopulations of antibodies that are specific for proPSA, particularlydifferent forms of proPSA. Because of the minor structural differencesbetween pPSA and PSA, the development of pPSA-specific mAbs has beenextremely difficult in the past. Mature PSA contains at least 6 majorantigenic epitopes¹¹ that induce a strong immune response in mice, andobscures the development of pPSA recognition. In the literature, effortsspecifically designed to generate pPSA mAbs have yielded no suitablemAbs¹². In the present invention, due to the successful expression ofchimeric pPSA protein, antibodies specific for pPSA may be generated byutilizing purified pPSA peptides and thus minimizing an interfering PSAimmune response. In addition, the present invention also focuses ondeveloping antibodies that detect different truncated pPSA forms.

[0009] Thus, one aspect of the present invention provides an antibody,preferably a monoclonal antibody, which specifically binds to proPSA.Antibodies to the various inactive precursor forms of proPSA, including,but not limited to [−2], [−4]pPSA, [−5]pPSA and [−7]pPSA, are alsocontemplated. Antibodies of the present invention are not only specificfor the pPSA region of the pPSA protein, but also capable of detectingthe even more subtle differences between [−7], [−4] and [−2]pPSA forms.

[0010] The present invention also encompasses a method for detectingproPSA in a human tissue or physiological fluid. This aspect of theinvention is based on the discovery that proPSA exists stably inbiological fluid as part of free PSA and may serve as a useful markerfor prostate cancer. Specifically, several inactive precursor forms ofPSA, such as, but not limited to [−2], [−4] and [−7]pPSA forms, havebeen identified and detected in serum. In accordance with the presentinvention, the identified precursor forms of PSA do not form a complexwith ACT and exist as stable and free PSA in serum. The measurement ofthese inactive precursor forms of PSA may provide important informationregarding the detection, monitoring and staging of prostate cancer.

[0011] Therefore, proPSA polypeptides, as well as variants and subunitsthereof, produced by the methods of the present invention can be used toproduce populations of antibodies that, in turn, can be used as thebasis for direct or competitive assays to detect and quantify proPSApolypeptides (or “protein”) in samples derived from physiologicalfluids, such as seminal fluid, blood or serum; tissues, such as prostatecarcinomas; or cells, such as prostate cells.

[0012] Direct and competitive assays to detect proPSA are also includedwithin the scope of the present invention. A method for detecting proPSAin a sample of human physiological fluid is described which includesproviding purified antibodies to pPSA, contacting the antibodies withthe sample to allow formation of complexes between the antibodies andpPSA, and determining the presence or amount of pPSA complexed with theantibodies.

[0013] Using the antibodies and immunoassays of the present invention,it is discovered that different pPSA forms exist in serum, and that the[−2]pPSA is the most prevalent form. It is also a surprise discovery ofthe present invention that [−2]pPSA comprises a significant percentageof the free PSA in prostate cancer serum.

[0014] Accordingly, based on the discoveries of the present invention,one aspect of the present invention provides diagnostic methods fordetecting and/or determining the presence of prostate cancer in asubject, or for distinguishing prostate cancer from non-cancer benigndisease in a subject. In accordance with embodiments of the presentinvention, such a method includes the steps of determining the amount ofpPSA contained in a sample of the subject, and correlating the amount ofpPSA to the presence of prostate cancer in the subject.

[0015] Kits for detecting or distinguishing prostate cancer from benigndisease are also included as embodiments of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0016]FIG. 1 is a schematic representation of PSA expression vector,pGTD-PSA.

[0017]FIG. 2 depicts the expression of PSA by AV12-PSA#8 cells. Serumcontaining spent media of AV12-PSA#8 cells was harvested each day for6sixconsecutive days. PSA concentration was measured using Tandem®-MPPSA assay. Viable cells were counted each day using trypan blue.

[0018]FIG. 3 shows chromatography profiles of different proteins. PanelA, hydrophobic interaction chromatography profile of the pPSA formspurified from the spent media of AV12 cells; and Panel B, relativeelution profile of hK2, PSA, and pPSA forms.

[0019]FIG. 4 depicts the HIC chromatographic profile of the differentforms of PSA. FIG. 4A shows the retention time for mature PSA and pPSA,including [−4]pPSA, [−5]pPSA and [−7]pPSA. FIG. 4B shows the retentiontime for PSA forms from a serum sample bound to a PSM773 affinitycolumn.

[0020]FIG. 5 depicts the chromatographic profile for a mixture ofpurified mature PSA and pPSA. FIG. 5A shows the chromatographic profilefor the protein mixture without the addition of ACT. FIG. 5B shows thechromatographic profile for the same protein mixture after incubationwith ACT for two hours at 37° C.

[0021]FIG. 6 are Western blots and immunoassay analyses of the PSA formspurified from the serum of prostate cancer biopsy-positive andbiopsy-negative men: Panel A, total serum PSA prior to purification;Panel B, % free PSA in the serum prior to purification; Panel C, Westernblot detection of [−2]pPSA; and Panel D, Western blot detection of [−4]plus [−7]pPSA.

[0022]FIG. 7 shows the results of immunohistochemical staining ofprostate tissues for [−2]pPSA.

[0023]FIG. 8 is a dot plot that shows the immno-assay results of 52 menwith clinical BPH and 92 controls.

[0024]FIG. 9 shows the ratio of [−2]pPSA/BPSA in biopsy-positive (i.e.,cancer) and biopsy-negative samples contained essentially the sameamount of total PSA in the serum.

[0025]FIG. 10 shows the relative activation rates of [−2]pPSA, [−4]pPSA,and [−5/−7]pPSA by hK2.

[0026]FIG. 11 is a hydrophobic interaction chromatographic profileshowing that [−2]pPSA was not activated to PSA after 5 h incubation withhK2 (panel A). The [−4]pPSA (panel B) and [−5/−7]pPSA were converted toPSA.

[0027]FIG. 12 shows the results of immunoassay analysis of 3 differentpPSA mAbs showing a high specificity for [−7]pPSA and no significantspecificity for mature PSA.

[0028]FIG. 13 are Western blots of different mature and pro PSA formsprobed with different mAbs that demonstrates specificity for [−2], [−4]and [−7]pPSA.

[0029]FIG. 14 shows an immunoassay of 3 different mAbs, PS2X373, PS2V276and PS2P446, showing their relative reactivities towards [−2]pPSA,[−4]pPSA, [−4/−7]pPSA and mature PSA.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0030] The identification of inactive precursor forms of PSA in serumsuggests that measuring serum concentrations of proPSA can be useful inthe detection and monitoring of prostate cancer. In order to discern thesteps involved in the biosynthesis of PSA and the activation of proPSAto mature PSA, the expression of PSA in mammalian cells is necessary.The details of expressing PSA in mammalian cells are provided in theco-pending U.S. patent application, Ser. Nos. 09/251,686 and 09/302,965,the content of which is incorporated herein in its entirety byreference.

[0031] As used herein, the terms “PSA” and “PSA polypeptide” are usedinterchangeably and include recombinant prepro, pro, and mature PSApolypeptides. The terms “proPSA, ” “pPSA, ” “proPSA polypeptide”, and“pPSA polypeptide” are used interchangeably and preferably encompass allinactive precursor forms of PSA, including, but not limited to,[−2]proPSA, [−4]proPSA, [−7]proPSA, and [−5]proPSA.

[0032] As used herein, “chimeric” means that a vector comprises DNA fromat least two different species, or comprises DNA from the same species,which is linked or associated in a manner which does not occur in the“native” or wild type of the species.

[0033] “Control sequences” is defined to mean DNA sequences necessaryfor the expression of an operably linked coding sequence in a particularhost organism. The control sequences that are suitable for prokaryoticcells, for example, include a promoter and, optionally, an operatorsequence and a ribosome binding site. Eukaryotic cells are known toutilize promoters, polyadenylation signals and enhancers.

[0034] “Operably linked” means that the nucleic acids are placed in afunctional relationship with another nucleic acid sequence. For example,DNA for a presequence or secretory leader is operably linked to DNA fora polypeptide if it is expressed as a preprotein that participates inthe secretion of the polypeptide; a promoter or enhancer is operablylinked to a coding sequence if it affects the transcription of thesequence; or a ribosome binding site is operably linked to a codingsequence if it is positioned so as to facilitate translation. Generally,“operably linked” means that the DNA sequences being linked arecontiguous and, in the case of a secretory leader, contiguous and inreading phase. However, enhancers do not have to be contiguous. Linkingis accomplished by ligation at convenient restriction sites. If suchsites do not exist, the synthetic oligonucleotide adaptors or linkersare used in accord with conventional practice.

[0035] The general methods for constructing recombinant DNA which cantransform target cells are well known to those skilled in the art, andthe same compositions and methods of construction may be utilized toproduce the DNA useful herein. For example, J. Sambrook et al.,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press (2ded., 1989) provides suitable methods of construction.

[0036] The recombinant DNA can be readily introduced into the targetcells by transfection with an expression vector comprising cDNA encodingPSA, for example, by the modified calcium phosphate precipitationprocedure of C. Chen et al., Mol. Cell Biol., 7, 2745 (1987).Transfection can also be accomplished by lipofection, using commerciallyavailable kits, e.g., those provided by BRL Life Technologies, Inc.

[0037] Suitable host cells for the expression of PSA are derived frommulticellular organisms. Such host cells are capable of complexprocessing and glycosylation activities. However, mammalian cells arethe preferred host for expression of mammalian protein, since thesecells modify and process the recombinant protein in a manner closelyrelated to the natural host of the protein. In principle, any highereukaryotic cell culture can be employed in the practice of theinvention, whether from vertebrate or invertebrate culture. Examples ofinvertebrate cells include plant and insect cells. Numerous baculoviralstrains and variants and corresponding permissive insect host cells havebeen identified.

[0038] “Polymerase chain reaction,” or “PCR,” refers to a procedure ortechnique in which amounts of a preselected fragment of nucleic acid,RNA and/or DNA, are amplified as described in U.S. Pat. No. 4,683,195.Generally, sequence information from the ends of the region of interestor beyond are employed to design oligonucleotide primers. These primerswill be identical or similar in sequence to opposite strands of thetemplate to be amplified. PCR can be used to amplify specific RNAsequences, specific DNA sequences from total genomic DNA, and cDNAtranscribed from total cellular RNA, bacteriophage or plasmid sequences,and the like. See, generally, Mullis et al., Cold Spring Harbor Symp.Quant. Biol., 51, 263 (1987); Erlich, ed., PCR Technology (StocktonPress, NY, 1989).

[0039] When a PSA polypeptide is expressed in a recombinant cell otherthan one of human origin, the PSA polypeptide is completely free ofproteins or polypeptides of human origin. However, it is necessary topurify PSA polypeptides from recombinant cell proteins or polypeptidesto obtain preparations that are substantially homogeneous as to PSApolypeptides. For example, the culture medium or lysate can becentrifuged to remove particulate cell debris. The membrane and solubleprotein fractions are then separated. The PSA polypeptide may then bepurified from the soluble protein fraction and, if necessary, from themembrane fraction of the culture lysate. The PSA polypeptide can then bepurified from contaminant soluble proteins and polypeptides byfractionation on immunoaffinity or ion exchange columns; ethanolprecipitation; reverse phase HPLC; chromatography on silica or on ananion exchange resin such as DEAE; chromatofocusing; SDS-PAGE; ammoniumsulfate precipitation; gel filtration using, for example, SEPHADEX G-75;or ligand affinity chromatography.

[0040] Once isolated, and in accordance with an embodiment of thepresent invention, a proPSA polypeptide or peptide corresponding to theproPSA region can be used to produce anti-pPSA antibodies. The proPSApolypeptides used to generate antibodies in accordance with theinvention include, but are not limited to, -7, -5, -4 and -2 proPSA.Peptides corresponding to the proPSA region can also be used to generateanti-pPSA antibodies and include all peptides which contain any portionof the pro region of the pPSA polypeptide. These peptides preferablycontain about 8 to 15 amino acids and comprise an immunogenic epitope.

[0041] In accordance with one embodiment of the present invention, propeptide SRIVGGWECEK may be used to generate antibodies for [−2]proPSA.Pro peptide ILSRIVGGWECEK may be used to generate antibodies for[4]proPSA. The purified recombinant protein consisting of the PSA preproleader peptide may be used to generate antibodies for [−7]proPSA.

[0042] In accordance with the present invention, an antibody whichconsists essentially of pooled monoclonal antibodies with differentepitopic specificities, as well as distinct monoclonal antibodypreparations, is provided. Monoclonal antibodies against purified pPSA(total protein) or the above peptides can be prepared using knownhybridoma cell culture techniques, for example, as described by E.Harlow et al., Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory, 1988. In general, this method involves preparing anantibody-producing fused cell line, e.g., of primary spleen cells fusedwith a compatible continuous line of myeloma cells, and growing thefused cells either in mass culture or in an animal species from whichthe myeloma cell line used was derived or is compatible. Such antibodiesoffer many advantages in comparison to those produced by the inoculationof animals, as they are highly specific and sensitive and relatively“pure” immunochemically. Immunologically active fragments of antibodiesare also within the scope of the present invention, e.g., the f(ab)fragment, as are partially humanized monoclonal antibodies.

[0043] If desired, polyclonal antibodies can be further purified, forexample, by binding to an elution from a matrix to which a polypeptide,or a peptide to which the antibodies were raised, is bound. Thoseskilled in the art will know of various techniques common in theimmunology arts for purification and/or concentration of polyclonalantibodies, as well as monoclonal antibodies. (Se, for example, Coliganet al, Unit 9, Current Protocols in Immunology, Wiley Interscience,1991, incorporated by reference.)

[0044] The term “antibody” as used in this invention includes intactmolecules as well as fragments thereof, such as Fab, F(ab′)₂, and Fv,which are capable of binding the epitopic determinant. These antibodyfragments retain some ability to selectively bind with their antigen orreceptor.

[0045] Accordingly, one aspect of the present invention provides anantibody that specifically binds to proPSA of the present invention. Theterm “specifically immunoreactive or specifically binds to” as usedherein indicates that the antibodies of the present invention recognizeand bind to antigenic determinants or epitopes that are unique toproPSA, and are not found on mature PSA. Examples of monoclonalantibodies that specifically bind to proPSA include, but are not limitedto, PS2P206, PS2P309, PS2P446, PS2P031, PS2P401, PS2P167, PS2P125,PS2P134, PS2X094, PS2X373, PS2X199, PS2X458, PS2X572, PS2V411, andPS2V476. For example, monoclonal antibody PS2P446 is specific for [−7]and/[−4]pPSA by Western blot analysis, though specific for [−7]pPSA byimmunoassay. PS2X373 is specific for [−2]pPSA. PS2V476 is specific for[−4]pPSA.

[0046] Antibodies of the present invention may be used for detecting anddetermining the presence and amount of proPSA in a sample. They may alsobe used for detecting and determining the presence and amount ofdifferent forms of proPSA in a sample. In accordance with the presentinvention, the proPSA may be detected in patient tissue samples byimmunohistochemical methods and/or in patient fluid samples by in vitroimmunoassay procedures.

[0047] Immunohistochemical methods for the detection of antigens inpatient tissue specimens are well-known in the art and need not bedescribed in detail herein. For example, methods for theimmunohistochemical detection of antigens are generally described inTaylor, Arch. Pathol. Lab. Med. 102:113 (1978). Briefly, in the contextof the present invention, a tissue specimen obtained from a patientsuspected of having a prostate-related problem is contacted with anantibody, preferably a monoclonal antibody, recognizing proPSA. The siteat which the antibody is bound is thereafter determined by selectivestaining of the tissue specimen by standard immunohistochemicalprocedures. In one embodiment of the present invention, the tissuespecimen is a tissue specimen obtained from the prostate of a patient.The prostate tissue may be a normal or benign prostate tissue, a cancerprostate tissue, or a benign prostatic hyperplasia tissue.

[0048] Similarly, the general methods of the in vitro detection ofantigenic substances in patient fluid samples by immunoassay proceduresare also well-known in the art and require no repetition herein. Forexample, immunoassay procedures are generally described in Paterson etal., Int. J. Can. 37:659 (1986) and Burchell et al., Int. J. Can. 34:763(1984). According to one embodiment of the present invention, animmunoassay for detecting proPSA in a biological sample comprises thesteps of: (a) contacting an antibody that specifically binds to proPSAwith the sample under a condition that allows a formation of a binarycomplex comprising the proPSA and the antibody ; and (b) detecting anddetermining the amount of the complex.

[0049] For the purpose of the present invention, the biological samplecan be any human physiological fluid sample that contains proPSA of thepresent invention. Examples of the human physiological fluid sampleinclude, but are not limited to, blood, serum, seminal fluid, urine, andplasma.

[0050] For the purpose of the present invention, both monoclonalantibodies and polyclonal antibodies may be used as long as suchantibodies possess the requisite specificity for the antigen provided bythe present invention. Preferably, monoclonal antibodies are used.

[0051] Monoclonal antibodies can be utilized in a liquid phase or boundto a solid phase carrier. Monoclonal antibodies can be bound to manydifferent carriers and used to determine the proPSA of the presentinvention. Examples of well-known carriers include glass, polystyrene,polypropylene, polyethylene, dextran, nylon, amylases, natural andmodified celluloses, polyacrylamides, agaroses, and magnetites. Thenature of the carrier can be either soluble or insoluble for purposes ofthe invention. Examples of insoluble carriers include, but are notlimited to, a bead and a microtiter plate. Those skilled in the art willknow of other suitable carriers for binding monoclonal antibodies orwill be able to ascertain such under routine experimentation.

[0052] In addition, the monoclonal antibodies in these immunoassays canbe detectably labeled in various ways. For example, monoclonalantibodies of the present invention can be coupled to low molecularweight haptens. These haptens can then be specifically detected by meansof a second reaction. For example, it is common to use such haptens asbiotin, which reacts with avidin, or dinitrophenyl, pyridoxal andfluorescein, which can react with specific antihapten antibodies. Inaddition, monoclonal antibodies of the present invention can also becoupled with a detectable label such as an enzyme, radioactive isotope,fluorescent compound or metal, chemiluminescent compound, orbioluminescent compound. Furthermore, the binding of these labels to thedesired molecule can be done using standard techniques common to thoseof ordinary skill in the art.

[0053] One of the ways in which the antibody can be detectably labeledis by linking it to an enzyme. This enzyme, in turn, when later exposedto its substrate, will react with the substrate in such a manner as toproduce a chemical moiety which can be detected by, for example, aspectrophotometric or fluorometric means (ELISA system). Examples ofenzymes that can be used as detectable labels are horseradishperoxidase, malate dehydrogenase, staphylococcal nuclease,delta-5-steroid isomerase, yeast alcohol dehydrogenase,alpha-glycerophosphate dehydrogenase, triose phosphate isomerase,alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase,ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase,glucoaamylase, and acetylcholine esterase.

[0054] For increased sensitivity in the ELISA system, the proceduresdescribed can be modified using biotinylated antibodies reacting withavidin-peroxidase conjugates.

[0055] The amount of antigen can also be determined by labeling theantibody with a radioactive isotope. The presence of the radioactiveisotope would then be determined by such means as the use of a gammacounter or a scintillation counter. Isotopes which are particularlyuseful are ³H, ¹²⁵I, ¹²³I, ³²P, ³⁵S, ¹⁴C, ⁵¹Cr, ³⁶Cl, ⁵⁷Co, ⁵⁸Co, ⁵⁹Fe,⁷⁵Se, ¹¹¹N, ⁹⁹mTc, ⁶⁷Ga, and 90Y.

[0056] Determination of the antigen is also possible by labeling theantibody with a fluorescent compound. When the fluorescently labeledmolecule is exposed to light of the proper wave length, its presence canthen be detected due to fluorescence of the dye. Among the mostimportant fluorescent labeling compounds are fluorescein isothiocyanate,rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde,and fluorescamine.

[0057] Fluorescence-emitting metal atoms, such as Eu (europium), andother lanthanides, can also be used. These can be attached to thedesired molecule by means of metal-chelating groups, such as DTPA orEDTA.

[0058] Another way in which the antibody can be detectably labeled is bycoupling it to a chemiluminescent compound. The presence of thechemiluminescent-tagged immunoglobulin is then determined by detectingthe presence of luminescence that arises during the course of a chemicalreaction. Examples of particularly useful chemiluminescent labelingcompounds are luminol, isoluminol, aromatic acridinium ester, imidazole,acridinium salt, and oxalate ester.

[0059] Likewise, a bioluminescent compound may also be used as a label.Bioluminescence is a special type of chemiluminescence which is found inbiological systems in which a catalytic protein increases the efficiencyof the chemiluminescent reaction. The presence of a bioluminescentmolecule would be determined by detecting the presence of luminescence.Important bioluminescent compounds for purposes of labeling areluciferin, luciferase, and aequorin.

[0060] Qualitative and/or quantitative determinations of proPSA of thepresent invention in a sample may be accomplished by competitive ornon-competitive immunoassay procedures in either a direct or indirectformat. Examples of such immunoassays are the radioimmunoassay (RIA) andthe sandwich (immunometric) assay. Detection of the antigens using themonoclonal antibodies of the present invention can be done utilizingimmunoassays which are run in either the forward, reverse, orsimultaneous modes, including immunohistochemical assays onphysiological samples. Those skilled in the art will know, or canreadily discern, other immunoassay formats without undueexperimentation.

[0061] The terms “immunometric assay” or “sandwich immunoassay” includea simultaneous sandwich, forward sandwich, and reverse sandwichimmunoassay. These terms are well understood by those skilled in theart. Those skilled in the art will also appreciate that antibodiesaccording to the present invention will be useful in other variationsand forms of assays which are presently known or which may be developedin the future. These are intended to be included within the scope of thepresent invention.

[0062] As well as being useful as an antigen to produce the presentanti-pPSA antibodies, the isolated pPSA polypeptide produced inaccordance with a method of the present invention and its antigenicallyactive variants, derivatives, and fragments thereof can be used inassays for proPSA in samples derived from biological materials suspectedof containing proPSA or anti-proPSA antibodies.

[0063] A useful immunoassay which can be practiced in accordance with anembodiment of the present invention is the two-antibody sandwichtechnique. These assays are used primarily to determine the antigenconcentration in unknown samples. Two-antibody assays are quick andaccurate, and if a source of pure antigen (in this case, proPSA) isavailable, the assays can be used to determine the absolute amounts ofantigen in unknown samples. The assay requires two antibodies that bindto non-overlapping epitopes on the antigen. Either two monoclonalantibodies that recognize discrete sites or one batch ofaffinity-purified polyclonal antibodies can be used.

[0064] In a two-antibody assay, one antibody is purified and bound to asolid phase. Any solid phase can be used; however, for mostapplications, a PVC microtiter plate is preferred. The bound antibody(to a well of a microtiter plate, for example) is unlabeled and isreferred to as the “capture antibody.” The amount of antibody to be usedwill depend on the individual assay, but an amount of about 1 μg/wellgenerally gives maximal binding. Higher or lower amounts of captureantibody can also be used. The wells can then be washed and sample addedto the wells to allow the antigen (in this case, pPSA) in the testsolution to bind to the solid phase. Unbound proteins can be removed bywashing and a labeled second antibody can be added. Alternatively, thesample and the second labeled antibody can be added simultaneously.After washing, the assay can be quantitated by measuring the amount oflabeled second antibody that is bound to the solid phase. A mostpreferred embodiment of the present invention utilizes a monoclonalantibody as the first unlabeled antibody and a monoclonal antibody asthe second labeled antibody. The detection method used to quantitate theamount of bound labeled antibody depends on the label used. Antibodiescan be labeled conveniently with iodine, enzymes, or biotin.Calorimetric or other detection methods can be used.

[0065] The proPSA polypeptides of the present invention can beimmobilized and used as “capture antigens” to bind and immobilizeanti-pPSA antibodies from a sample to be assayed for anti-pPSAantibodies. The bivalent complex of proPSA polypeptides and anti-pPSAantibodies is then detected, e.g., in the case of human physiologicalmaterial, by reacting it with an anti-human IgG antibody which comprisesa detectable label or a binding site for a detectable label. In thelatter case, the binding site is itself reacted with a compound specificfor the binding site, which itself comprises a detectable label. Usefuldetectable labels include enzymes, radio labels, or fluorescent labels.The resultant ternary or quaternary complex can then be detected and/orquantified via the detectable label, i.e., via an enzyme-substratecolor-forming reaction, radio emission, agglomeration, and the like.

[0066] Alternatively, the proPSA polypeptide can be labeled with adetectable label, such as via one or more radio labeled peptidylresidues, and can be used to compete with endogenous proPSA for bindingto anti-proPSA antibodies, i.e., as a capture antigen to bind toanti-proPSA antibodies in a sample of a physiological fluid via variouscompetitive immunoassay formats. For example, a competitive immunoassayfor proPSA which uses immobilized anti-proPSA antibodies is carried outby:

[0067] (a) providing an amount of proPSA specific antibodies attached toa solid surface;

[0068] (b) mixing the sample of physiological fluid to be tested with aknown amount of proPSA polypeptide which comprises a detectable label toproduce a mixed sample;

[0069] (c) contacting said antibodies with said mixed sample for asufficient time to allow immunological reactions to occur between saidantibodies and said proPSA to form an antibody-proPSA complex, andbetween said antibodies and said labeled polypeptide to form anantibody-labeled polypeptide complex;

[0070] (d) separating the antibodies which are bound to proPSA andantibodies bound to the labeled polypeptide from the mixed sample;

[0071] (e) detecting or determining the presence or amount of labeledpolypeptide either bound to the antibodies on the solid surface orremaining in the mixed sample; and

[0072] (f) determining from the result in step (e) the presence oramount of said proPSA in said sample.

[0073] The immunoassays of the present invention may be used to detectpPSA in human physiological samples, such as serum and tissue, for thepurpose of detecting and monitoring prostate cancer. The assays of thepresent invention may also be used to distinguish prostate cancer frombenign prostate disease, such as benign prostatic hyperplasia.

[0074] It is a discovery of the present invention that pPSA, i.e., [−2]and [−4]pPSA, are present in prostate cancer serum and tissues.Particularly, it is a surprise discovery of the present invention that[−2]pPSA is not only present in prostate cancer serum, but is found moreconsistently, and comprises a major percentage of the free PSA. Moreimportantly, it is a discovery of the present invention that the majorpercentage of the free PSA is [−2]pPSA in the prostate cancer serum ofmen with total PSA values from 6-24 ng/ml. Therefore, pPSA, particularly[−2]pPSA, represents a significant and relevant isoform of PSA for thestudy of prostate cancer. It is in the range below 10 ng/ml where it ismost difficult to discriminate prostate cancer from benign conditionssuch as BPH since both conditions release PSA into the serum ^(13,14).In addition, while the [−4] and [−7]pPSA forms appear to be absent insome samples of the limited serum group tested, it is possible each ofthese forms may show specific utility in a larger group. Therefore,[−2]pPSA, or other pPSA forms could offer the greatest diagnostic valueas the percentage of total PSA, as a percentage of the free PSA, or asan independent indicator where pPSA levels above a certain thresholdhave a high statistical correlation with cancer. In the latter case, thepresence of pPSA could offer diagnostic value in serum containing anymeasurable level of pPSA, and especially in serum with less than 4 ng/mltotal PSA.

[0075] Accordingly, the present invention provides a diagnostic methodfor detecting or determining the presence of the prostate cancer in asubject. In accordance with the embodiments of the present invention, adiagnostic method of the present invention may include the steps ofdetermining the amount of pPSA in a sample of the subject, andcorrelating the amount of the pPSA to the presence of prostate cancer.In accordance with one embodiment of the present invention, pPSA may be[−2], [−4], and/or [−7]pPSA. In one embodiment, pPSA is [−2]pPSA.

[0076] The amount of pPSA may be measured by any method described hereinor known in the art, or later developed, as long as they are capable ofmaking such a measurement. In accordance with one embodiment of thepresent invention, pPSA contained in a sample may be measured by amethod including the steps of contacting an antibody that binds withsufficient specificity to proPSA in the sample under a condition thatallows the formation of a binary complex comprising the proPSA and theantibody, and detecting and determining the amount of the complex.

[0077] For the purpose of the present invention, the binding of anantibody to pPSA is sufficiently specific if the antibody can bepractically used to achieve the discrimination between pPSA and otherforms of PSA, and therefore to allow the detection and determination ofthe amount of pPSA in a given application and format. Examples of suchantibodies include, but not limited to, antibodies that are specific forpPSA, and antibodies that preferentially bind to pPSA. In addition, notonly antibodies specifically or preferentially bind to a particularform, forms of pPSA, but also antibodies that specifically orpreferentially bind to all forms of pPSA are contemplated. An example ofsuch an antibody that specifically or preferentially binds to all formsof pPSA would be an antibody that detects either [−1] or [−2]pPSA butalso detects pPSA containing additional pro leader amino acids from [−3]through [−7].

[0078] The term “preferentially bind to” as used herein indicates thatthe antibody binds to pPSA to a greater extent than binding to otherforms of PSA. The degree or extent to which these antibodies recognizeproPSA better than other PSA forms will depend upon the specificapplication and format employed. The evaluation and selection ofantibodies with preferential binding is well known to those skilled inthe art and is a routine part of development of any immunoassay. Theterm “other forms of PSA” as used herein include any forms of PSA thatare not pPSA, such as other clipped or non-clipped mature forms of PSA.Examples of antibodies that preferentially bind to pPSA include, but arenot limited to PS1Z134, PS1Z120, PS1Z125, and PS1Z80.

[0079] In addition, any equivalents of the above discussed antibodiesmay also be used for measuring the amount of pPSA. For the purpose ofthe present invention, any molecular species, known or developed later,capable of binding to pPSA with sufficient specificity will beconsidered as equivalents of the antibodies, and therefore may be usedfor measuring the amount of pPSA. Such equivalents of the antibodies maybe selected by methods known in the art. For example, any known bindingassays may be used to determine the binding activity of any givenmolecule. . Examples of potential molecular species include, but are notlimited to, antigen-binding fragments derived from antibodies, andaptamers.

[0080] In accordance with embodiments of the present invention, theantibody is an antibody specific for pPSA. For example, any antibodiesdescribed herein that are specific for pPSA may be used in thediagnostic method of the present invention. In accordance with anotherembodiment of the present invention, the antibody is an antibody thatpreferentially recognize pPSA. Examples of such antibodies includePS1Z134, PS1Z120, PS1Z125, and PS1Z80.

[0081] For the purpose of the present invention, the amount of pPSAdetected in a sample of a patient may be correlated to the presence ofprostate cancer in any way that generates diagnostic value fordetermining the presence of prostate cancer. For example, the amount ofpPSA contained in a sample may be compared to the total PSA, the freePSA, the BPSA, or hK2 of a sample. The comparison, i.e., a mathematicalcombination, such as a ratio with other forms of PSA or hK2, or theamount of pPSA alone may be used as an indicator where pPSA levels abovea pre-determined threshold have a high statistical correlation withcancer. For example, the amount of pPSA may be compared to other formsof PSA or hK2 to generate a numerical result, wherein a result above apre-determined value is an indication of the presence of prostatecancer. For the purpose of the present invention, the term “mathematicalcombination” includes any mathematical combination that may generate anumerical result for determining the presence of prostate cancer. Whilea ratio may be the simplest mathematical combination, other morecomplicated forms such as polynomials, log-logic functions andartificial neural networks can also be used. These forms and others arewell known to mathematicians and biostatisticians.

[0082] In view of the teaching of the present invention, one skilled inthe art through routine experimentation can readily determine thecut-off values (the threshold) or other analytical parameters necessaryto use the above-discussed numerical results such as a ratio or theamount of pPSA alone as a marker in determining the presence of prostatecancer. For example, one may compare the above-discussed ratio or pPSAin individuals diagnosed with prostate cancer with individuals that donot have prostate cancer or have BPH to determine the cut-off valueswith required specificity and sensitivity. Then the ratio or the amountof pPSA of a sample may be compared to the pre-determined cut-off valuefor determining the presence of prostate cancer in a subject, whereinthe higher level of pPSA may be an indication of prostate cancer. Themethod of determining the cut-off value with required specificity andsensitivity are well known in the art, and need not be repeated³²⁻³⁹.

[0083] In addition, it is a discovery of the present invention thatpPSA, particularly [−2]pPSA is elevated in the serum and the tissues ofprostate cancer patients when compared to its amount in the serum or thetissues of men with benign prostateic disease such as BPH. Therefore,the above-discussed ratio or the amount of the pPSA independently mayalso be used to determine whether a subject has a greater likelihood ofprostate cancer rather than benign disease such as BPH. For example, onemay examine the levels of pPSA in patients diagnosed with prostatecancer and in patients diagnosed with benign disease to determine acut-off value. Then the ratio or amount of pPSA of an unknown patientsample may be compared to the pre-determined cut-off value, wherein thehigher level of pPSA compared to the pre-determined cut-off value is anindication of prostate cancer.

[0084] Methods of measuring total PSA and free PSA are well-known in theart, and therefore will not be repeated herein. For the purpose of thepresent invention the term “total PSA” refers to total immunologicallydetectable PSA. Immunologically detectable PSA is generally the sum ofthe free uncomplexed PSA and the complexed PSA. Complexed PSA isprimarily composed of PSA-ACT with lesser amounts of complexes withinhibitors other than ACT. Total PSA is measurable by commerciallyavailable assays such as the Hybritech® PSA assay for the ACCESS®immunoassay system. The term “free PSA” as used herein refers toenzymatically inactive PSA that circulates in blood unbound to anyprotease inhibitor. Free PSA is measurable by commercially availableassays such as the Hybritech® free PSA assay for the ACCESS® immunoassaysystem. BPSA and methods of measuring BPSA are fully described in thecommonly owned co-pending U.S. patent application Ser. Nos. 09/303,208and 09/303,339, the relevant content of which is incorporated herein inits entirety by reference. Briefly, BPSA refers to a form of PSA thatcomprises at least one clip at Lys 182 of the amino acid sequence of amature form of PSA. In other words, a BPSA of the present invention hasthe same amino acid sequence of a mature form of PSA, except that thepolypeptide chain of the PSA of the present invention has beenhydrolyzed between residues 182 and 183. It is discovered that whileproPSA is elevated in the tissues or the serum of a prostate cancerpatient, BPSA is elevated in the tissues or the serum of a patient withBPH. Therefore, by comparing proPSA with BPSA of a patient, one maydistinguish prostate cancer from BPH and determine the presence ofprostate cancer.

[0085] Antibodies of the present invention may also be used in adiagnostic kit for determining the presence of pPSA contained in asample. Accordingly, one aspect of the present invention provides a kitfor determining pPSA in a sample. The kit comprises a known amount of anantibody specific for pPSA. The kit may also comprise a solid support oradditional known amount of antibodies specific for pPSA. Antibodiescontained in the kit may be attached to a solid support, or may bedetectably labeled, or both respectively.

[0086] The invention is further described by reference to the followingdetailed examples.

EXAMPLES Materials and Methods

[0087] Expression Vector, Cell Line and Transfection

[0088] An 0.8-kb DNA fragment which has a nucleic acid sequence of SEQID. NO: 1, as set forth in FIG. 1, coding for entire ppPSA was clonedinto the Bcll site of pGT-d under the control of GBMT promoter ¹⁵resulting in the expression vector pGTD-PSA (FIG. 1). The orientationand the sequence of the insert was confirmed. AV12-664 (ATCC CRL 9595),cultured in DMEM (high glucose) and 10% FETAL CLONE (Hyclone, Logan,Utah), were transfected with pGTD-PSA using Lipofectamine (LifeTechnologies, Inc.). Transfected AV12-664 cells (AV12-PSA) were selectedin 400 nM methotrexate (Sigma Chemical Company). AV12-664 transfectedwith the empty vector (AV 12-PGTD) were also selected in a similarmaimer for use as a negative control. Single cell clones were isolated.Viability of cells was assessed by trypan blue dye exclusion.

[0089] Isolation of Recombinant pPSA Forms from Mammalian Cells

[0090] Recombinant PSA was expressed in mammalian AV12 and the spentmedia was passed over the PSA-specific mAb, PSM773. PSM773 has beenshown previously to have specificity for mature, clipped, and precursorforms of PSA¹⁶⁻¹⁸. The column was washed with 40 volumes of PBScontaining 0.1% reduced Triton-X100, and bound protein eluted with 100mM glycine pH 2.5, containing 200 mM sodium chloride. The eluant wasimmediately neutralized with 10% % vol/vol 1M Tris pH 8.0. The purifiedPSA contained no mature PSA but contained [−5/−7], [−4], and [−2]pPSAmolecular isoforms of pPSA that were purified by HIC-HPLC as describedbelow.

[0091] Immunoassay and SDS-PAGE of PSA

[0092] The concentration of PSA in serum, media, or purifiedpreparations was determined by Tandemt®-MP PSA and Tandem®-MP free PSAassays (Hybritech Incorporated, San Diego, Calif.). SDS-PAGE wasperformed using 4-20% gradient mini-gels (Invitrogen, Carlsbad, Calif.)under reducing or non-reducing conditions, as indicated. Samples wereelectroblotted onto nitrocellulose using standard procedures. PrimarypPSA mAbs were used at 5 ug/ml and incubated with the blots overnight at4° C. Blots were detected with a secondary antibody cocktail consistingof goat anti-mouse heavy and light chain-HRP 1:50,000 (JacksonImmunoresearch Laboratories, Inc., West Grove, Pa.). The immunoreactivesignals were detected by SuperSignal® West Dura Extended DurationSubstrate (Pierce Chemical Co., Rockford, Ill.), according to themanufacturer's instructions.

[0093] Assay for the Measurement of PSA Activity

[0094] Enzymatic activity of PSA was measured according to the procedurepublished by Christensson, A. et al., ¹⁹. Briefly, PSA preparations(either purified from seminal fluid or day 7 spent media of AV12-PSA#8cells) were incubated with 1 mM pNA-derivatized peptide chromogenicsubstrates (methoxysuccinyl-Arg-Pro-Tyr-pNA, S2586; Pharmacia Hepar,Inc.) in 200 mM Tris/5 mM EDTA (pH 8.0) at 37° C. The enzymatic activityof PSA was determined by hydrolysis of the peptide chromogenicsubstrates, leading to an increase in absorbance at 405 nm.

[0095] HIC-HPLC of PSA

[0096] High performance hydrophobic interaction chromatography(HIC-HPLC) was performed using a polypropylaspartamide column (PolyLC,distributed by Western Analytical, Temecula, Calif.). The column was4.6×250 mm in length with a 1000 Å pore size. Samples were applied in1.5 M ammonium sulfate and eluted with a gradient: Buffer A: 1.2 Msodium sulfate, 25 mM sodium phosphate, pH 6.3, and Buffer B: 50 mMsodium phosphate, 5% v/v 2-propanol. The gradient was 0-35 % B, 1 min,30-80% B, 12 min, then isocratic at 80% B for 2 min before equilibrationin Buffer A. High sensitivity peak detection was obtained with a VarianModel 9070 scanning fluorescence detector using an excitation of 232 nmand emission of 334 nm to detect the tryptophan residues in protein.

[0097] Purification and Detection of PPSA from Pooled Prostate CancerSerum

[0098] 75 mls of pooled human serum from prostate cancer patients withelevated PSA was obtained. Solid ammonium sulfate was added to the serumto make the final concentration 2M and then the sample was dialyzedversus 2 M ammonium sulfate for 16 hours at 4° C. The serum was thenclarified by centrifugation and the supernatant solution dialyzed threetimes (one hour each time) against 2 liters of 20 mM sodium phosphate,pH 7. The sample was then filtered through a 0.2μ membrane filter andpassed over a 0.5 ml affinity column at 1 ml/min. The affinity columnconsisted of the mAb PSM773 covalently bound to AMINOLINK (Pierce) resinat a concentration of 5 mg mAbs per ml of resin.

[0099] The affinity column was washed with 50 mls of PBS and the PSAeluted with 3×1 ml volumes of 100 mM glycine, 0.5 M sodium chloride, pH2.5. The eluant (3 mls) was neutralized with 300 μl of 1 M Tris, pH 8.Ammonium sulfate was added to the eluant to a final concentration of 2 Mand this sample was applied to an HPLC column to be resolved byhydrophobic interaction chromatography as described above.

[0100] Development of Monoclonal Antibodies to pPSA

[0101] mAbs to [−2] and [−4]pPSA were developed by mouse immunizationwith peptides attached to Keyhole limpet hemocyanin (Pierce Chemical Co.Rockford, Ill.). For [−2]pPSA, the pro peptide was SRIVGGWECEK, and for[−4]pPSA, the peptide was ILSRIVGGWECEK. Hybridomas were produced byusual methodologies²⁰ and the antibody clones selected by reactivity tothe respective peptide indicated above, and no reactivity to the controlpeptide for mature PSA, IVGGWECEK. The clones were further selected ontheir ability to recognize purified [−2] and [−4]pPSA protein on Westernblots. When SDS-PAGE was run under reducing conditions, PS2X373 showedabout 20% cross-reactivity to the mature PSA protein by Western blot.However, the cross-reactivity dropped to 5% or less under non-reducingconditions and so these conditions were used for the detection of[−2]pPSA in the FIG. 6C.

[0102] mAbs to full length [−7]pPSA were obtained by mouse immunizationwith purified recombinant chimeric protein consisting of the PSA preproleader peptide attached to human kallikrein 2^(21,22). Clones werescreened on their recognition of native recombinant pPSA, and norecognition of native mature PSA. These mAbs were found to recognizeonly [−7]pPSA by immunoassay, but to recognize both [−7] and [−4]pPSAproteins equivalently on Western blots.

[0103] Generation of Monoclonal Antibodies with Preferential selectivityfor pPSA

[0104] PSA was purified from the medium of AV12 by the use ofimmunoaffinity chromatography using the anti-PSA antibody PSM773 asdescribed above. The mice were immunized once with 50 ug of blockedimmunogen in CFA and twice with 25 :g of blocked immunogen in IFA. Thehybridoma culture supernatant was screened for reactivity against pPSA.Hybridomas were screened by adding 50 ul of culture supernatant wasadded to the wells of a streptavidin microplate (Wallac, Turku, Finland)and 50 ul of biotinylated pPSA at 100 ng/ml was also added. After one hrincubation the plate was washed with PBS/0.1% tween-20, then incubatedwith 50 ul per well of goat anti-mouse Ig horseradish peroxidase(1:10,000) diluted in PBS/1% BSA and 0.1% tween-20. After one hrincubation, the plate was washed and developed with OPD substrate. Todetermine the specificity of monoclonal antibodies, the reactivity of100 ng/ml pPSA and 100 ng/ml intact PSA was compared

[0105] Development of monoclonal antibodies to BPSA isoforms

[0106] Processed, filtered seminal plasma was diluted 1:10 in PBS andpassed over an immunoaffinity column with bound anti-PSA mAb, PSM773.The column was washed with 20 volumes of PBS containing 0.1% reducedTriton X100, and the PSA eluted with 100 mM glycine pH 2.5 containing200 mM sodium chloride. The purified PSA was applied to HIC-HPLC asdescribed previously ²³ and the 8 min BPSA peak and the 10 min PSA peakwere collected separately. The PSA from the 10 min peak was dialyzedinto 100 mM Tris, pH 8 and incubated with 1% w/w trypsin for 30 min at37° C. The trypsin in the mixture was inactivated by addition of a massof aprotinin equal to twice the added trypsin. The incubation mixturewas applied to HIC-HPLC and the resultant in vitro BPSA peak wascollected. A detailed description of this process may also be found inthe commonly owned co-pending U.S. patent application Ser. No.09/303,208 the relevant content of which is incorporated herein byreference.

[0107] The in vitro BPSA was used as an immunogen in mice using standardprotocols. Antibodies were selected on their ability to recognize theimmunogen in preference to PSA which did not contain the clips at Lys145 and Lys182. Using standard hybridoma technologies, the monoclonalantibody PS2E290, a BPSA specific mAb was developed. PS2E290 was used ina dual mAb immunoassay to detect BPSA in serum.

[0108] Immunoassay BPSA

[0109] The immunoassay we have developed for the measurement of BPSA isas follows. 50 ul of biotinylated anti-PSA Ab PSM 773 at 5 ug/ml inTandem PSA zero cal diluent is added to a EG&G Wallac strep-avidincoated microplate and allowed to react at room temperature for 1 hourwith shaking. The plate is then washed 5 times with Tandem E wash. 50 ulof Tandem PSA zero cal diluent is then added to the plate followed by 50ul of sera or antigen to be tested. The mixture is allowed to react atroom temperature for 2 hours as above. The plate is then washed 5 timeswith Tandem E wash. 100 ul of a 1 mA solution of PS2E 290—alkalinephosphatase conjugate is add to the plate. The mixture is allowed toreact at room temperature for 1 hour as above. The plate is then washed5 times with Tandem E wash. 100 ul of Sigma 4MU-p solution is added toeach well and allowed to react at room temperature. After 1 hour theplate is read on a EG&G Wallac Victor instrument.

[0110] Amino Acid Sequencing of PSA

[0111] N-terminal sequence of the samples was performed through 9 cycleson a PE-Applied Biosystems Model 492 amino acid sequencer (PE-AppliedBiosystems, Foster City, Calif.). Purified PSA and peaks collected byHIC-HPLC were applied directly to PVDF membranes using the Prosorbcartridges (PE-Applied Biosystems, Foster City, Calif.), washed 3 × with0.1 mL 0.1% trifluoroacetic acid, and applied to the Model 492sequencer.

EXAMPLE 1 Expression of pPSA in Mammalian Cells

[0112] The cDNA for PSA was cloned into the pGT-d vector under thecontrol of the GBMT promoter using an approach similar to the onedescribed for hK2 by Kumar et al. To study the expression of PSA, AV12cells were transfected with the pGTD-PSA expression vector. Cells wereselected in 400 nM methotrexate for 2-3 weeks, and single cell cloneswere analyzed for PSA expression using TANDEM®-MP PSA assay and onWestern blots using mAb PSM 773. Clone AV12-PSA#8 was selected based onits high expression levels of a PSA-immunoreactive band at ˜32 kDa.

[0113] To determine the PSA expression pattern in mammalian cells,samples of spent media from AV12-PSA#8 cells were collected for sixconsecutive days and analyzed using the TANDEMOR-MP PSA assay. FIG. 2shows that PSA was detected in spent media at day 1 and accumulatedto >91 μg/ml by day 6. Expression of PSA was higher during the log phaseof cell growth, indicating that a stable form of PSA is secreted by thecells as opposed to being released following cell death and lysis. Whenthe same samples were analyzed for free PSA, similar values wereobtained (data not shown) indicating that AV12-PSA#8 cells expressuncomplexed or free PSA.

[0114] To determine the identity of the protein that is secreted fromthe cells, the spent media from AV12-PSA#8 cells was collected andconcentrated. The PSA in the media was purified by affinitychromatography using PSM 773, a PSA-specific mAb. The recombinant PSAexpressed in mammalian AV12 cells was found to be secreted as pPSA asdescribed previously in the commonly owned co-pending patent applicationSer. Nos. 09/302,965, and 08/846,408. No measurable differences wereobserved between the PSA purified from day 1 or day 7 media. The pPSAwas resolved into three different molecular forms by HJC-HPLC, as shownin FIG. 3A. PSA is normally expressed with a 7 aa pro leader peptideconsisting of APLILSR but an N-terminal sequencing of the 3 peaksresolved by HIC-HPLC in FIG. 3A indicated truncated forms of pPSA. PeakA contained approximately equal levels of the 7 aa pro leader peptideAPLILSR [−7]pPSA) and a clipped, truncated 5 aa leader peptidecontaining LILSR ([−5]pPSA) that were not resolved from one another.Peak B contained the 4 aa pro leader peptide ILSR ([−4]pPSA). Peak Ccontained the 2 aa leader peptide, SR ([−2]pPSA), in addition to about30% of PSA missing the first 4 aa of the mature sequence. Pure [−2]pPSAcould be obtained by collecting the back half of this peak. The decreasein retention time of these proteins resulting from the incrementaltruncation of the pro leader peptide is most likely due toconformational changes in the pPSA protein and not due to the minorsurface changes induced by removal of the indicated amino acids.

[0115]FIG. 3A shows that the different forms of pPSA can be resolved andpurified form one another by HIC-HPLC. FIG. 3B is an overlay fromseveral individual runs and shows the purified pPSA forms in addition tomature PSA and another prostate specific kallikrein, hK2. Thus, a singlechromatographic run can separate and resolve all of the major forms ofPSA protein from one another.

EXAMPLE 2 Detection of pPSA in Pooled Human Prostate Cancer Serum

[0116] The presence of pPSA in human serum would indicate the following.First, that PSA is secreted as the pPSA form in human tissue and isconverted to mature PSA extracellularly. Second, that pPSA is stable inhuman serum and thus may be a useful diagnostic marker for pCa or BPH.We evaluated the presence of pPSA in human serum by first using affinitypurification to purify all forms of PSA present in a pool of humanserum. We next fractionated the eluted PSA forms on HPLC and identifiedeach PSA form based on its elution profile from the column. Thisanalysis indicated that pPSA is present in human serum.

[0117]FIG. 4A shows the RT of standards of the different forms of PSA.All forms were verified by amino acid sequencing. The [−7, −5]pPSA peakcontains approximately equal levels of [−7]pPSA and [−5]pPSA forms whichare not resolved from each other. FIG. 4B shows the profile of PSA formsfrom serum bound to the PSM773 affinity column as described above.Samples were collected in 0.5 ml fractions and assayed by TANDEM®-MPfree PSA assay (FPSA assay, Hybritech Incorporated). The fPSA assaydetects both pPSA and free (inactive) PSA. The minor peak at sevenminutes is due to the slight cross-reactivity of the FPSA assay to thePSA-ACT eluted from the affinity column. The actual level of PSA-ACT inthis sample is about ten times higher than the level of free PSA (datanot shown). The peaks at 10 minutes and 12 minutes correspond to maturePSA and [−4]pPSA, respectively. These data indicate that at least oneform of pPSA ([−4]pPSA) is present in human serum and, as judged by therelative peak areas, it makes up approximately 25% of the free oruncomplexed PSA in this pooled serum sample.

[0118] To confirm that the pro forms of PSA are not reactive with ACT, amixture of purified mature PSA, [−4]pPSA and [−7,−5]pPSA, were incubatedwith ACT. FIG. 5A (W/O ACT) shows the chromatographic profile of theprotein mixture without the addition of ACT. FIG. 5B (+ACT) shows thechromatographic profile of an identical amount of the same mixture afterincubation with ACT for two hours at 37° C. Only the mature PSA forms anACT complex. The [−4], [−5], and [−7] forms of pPSA did not form acomplex with ACT, as they showed no decrease in peak area. This data isconsistent with the [−4]pPSA in serum not being complexed with ACT.

EXAMPLE 3 Detection of Truncated pPSA Forms in Prostate Cancer Serum

[0119] Serum from several individual men were also analyzed by Westernblot using newly developed pPSA mAbs. PSA was purified from the serum ofmen who were biopsy-positive and biopsy-negative for prostate cancer.The serum PSA values of the 5 biopsy-positive men were 6, 9, 10, 18, and24 ng/ml. Three biopsy-negative men with 7, 10, and 12 ng/ml total PSAwere also analyzed. Since the free PSA represented only 10-20% of thetotal PSA, and it was not known what percentage of the free PSA might becomprised of pPSA forms, it was necessary to purify PSA from 100-200 mlsof serum in order to be assured of adequate detection sensitivity forWestern blot analysis. Total PSA was purified from the serum byimmunoaffinity chromatography using the anti-PSA mAb, PSM773, whichrecognizes all forms of free PSA and PSA bound to ACT. The recovery ofPSA ranged from 30-60% of the amounts calculated to be in the startingserum by immunoassay. FIG. 6 shows the Western blot and immunoassayanalysis of these samples. In panels A-D, all samples are shown in thesame order. The first 5 samples were the PCa samples, and the last 3samples were from biopsy-negative men. Panels A and B are the ng/ml oftotal PSA, and the % free PSA, respectively, in the serum prior toimmunoaffinity purification. Panel B shows that the % free PSA wasgenerally lower in the cancer samples compared to biopsy-negative orbenign serum, which is in agreement with the predicted trend that BPHsamples should contain higher % free PSA. It should be noted that the %free PSA measured in the purified PSA was unchanged from the originalserum values, confirming that the purification procedure had noselectivity for either free or complexed forms of PSA.

[0120] Panels C and D are the Western blots of the same samples inpanels A and B, probed with mAbs to [2]pPSA and [−4/−7]pPSA,respectively. In FIG. 6C, the blot was probed with PS2X373, which isspecific for [−2]pPSA. Each lane was loaded with 11 ng of free PSA, asdetermined by prior immunoassay. Lane 1 contained 10 ng of purified[−2]pPSA standard. FIG. 6C shows that the 5 cancer samples contained thehighest levels of [−2]pPSA. Comparing band intensity to the 10 ng[−2]pPSA standard in lane 1 indicates that nearly all of the free PSA inlane 2 is [−2]pPSA. [−2]pPSA was estimated to comprise about half of thefree PSA in lanes 3-5, and to be 25% in lane 6 (Table 1). Lane 7, thefirst biopsy-negative sample, contained approximately 20% pPSA. Theoriginal serum assay for lane 7 measured 12 ng/ml total PSA, compared to6 ng/ml in the cancer sample in lane 6 (panel A), though both containedcomparable % free PSA (panel B). The biopsy-negative samples in lanes 8and 9 contained only nominal levels of pPSA, perhaps 5-10%. Since thePS2X373 mAb had a minor cross-reactivity of approximately 5% with maturePSA under these Western blot conditions, it is possible that some or allof the apparent band in these lanes is due to PSA cross-reactivity.

[0121] In Panel D, 3 ng of free PSA was loaded in each lane and probedwith PS2P446 which had equal specificity for [−4]pPSA and [−7]pPSA onWestern blots. The results of this blot indicated that other forms ofpPSA were present in only 2/5 of the cancer samples. PS2P446 had nocross-reactivity with mature PSA, and so the faint pPSA bands seen in2/3 of the biopsy-negative samples indicated low levels of these pPSAforms. We had also developed mAbs with specificity for [−4]pPSA, and nocross-reactivity to other pro or mature forms of PSA, but these mAbs hada weak reactivity on blots, and consequently, a high background. WhilePS2V476 clearly indicated the presence of [−4]pPSA, quantificationestimates were not possible. It is therefore not clear what proportionof the band intensities in panel D are due to [−4]pPSA or to full length[−7]pPSA. It is, however, evident that neither form of pPSA is asconsistently present in the cancer serum as [−2]pPSA. In controlexperiments, female serum, passed over the immunoaffinity column andworked up identically with the male serum, showed no band of pPSA whenprobed with the above pPSA-specific mAbs.

EXAMPLE 4 Immunohistochemical Staining of pPSA in Prostate Tissue

[0122] We tested the pPSA mAbs for staining on prostate tissues. ThePS2P446 ([−7]/[−4]pPSA specificity) and PS2X373 ([−2]pPSA specificity)had similar staining intensities, while PS2V476 ([−4]pPSA specificity)did not work well for immunostaining. Both PS2X373 and PS2P446 showed agenerally similar staining pattern as indicated in FIG. 7A, thoughPS2X373 showed more intense staining in cancer secretions as shown inFIG. 7B. Cancer epithelium showed consistent epithelial staining in the9 cancers tested. PIN also showed consistent strong staining. Benigntissues stained less intensely than the cancer in general, though thesurrounding benign tissues were more variable. FIG. 7A demonstrates thattruncated pPSA forms can be detected in the epithelium of fixed prostatetissues, which provides further support that the truncated pPSA formsare not an artifactual result of tissue extraction, but are naturallypresent in prostate tissues. Since PS2X373 and PS2P446 recognizedifferent forms of pPSA, comparable staining by both mAbs (not shown)indicates that multiple forms of pPSA are present in prostate tissues.The more intense staining of the [−2]pPSA in cancer secretions (FIG. 7B)could explain the prevalence of this form in the cancer serum.

EXAMPLE 5 Immunoassay for BPSA in Human Serum

[0123] We have previously identified a BPH-associated form of PSA calledBPSA. A detailed description of this process may also be found in thecommonly owned co-pending U.S. patent application Ser. No. 09/303,208,the relevant content of which is incorporated herein by reference. BPSAis found in prostate tissue containing BPH-nodules, but is notcorrelated with prostate cancer tissue. We have developed an immunoassaywith high specificity and sensitivity for BPSA as described in theMethods section. Serum from 52 men diagnosed with clinical benignprostatic hyperplasia (BPH) were assayed for BPSA by immunoassay. Inaddition, 92 men with no diagnosis of prostate disease were also assayedfor BPSA as a control group. FIG. 8 shows the assay results of 52 menwith clinical BPH and 92 controls. Men with BPH contained a median BPSAlevel of 116 pg/ml, while the median BPSA level was below the minimumdetectable limit of 60 pg/ml. These results demonstrate that BPSA ispresent in the serum of patients with clinically diagnosed BPH.

EXAMPLE 6 The Ratio of [−2]pPSA and BPSA in Human Serum

[0124] The patient samples in FIG. 6 were further analyzed for BPSA byimmunoassay. Table 1 shows the results of the [−2]pPSA and BPSAanalysis. The [−2]pPSA values were determined by densitometric analysisof the Western blot bands, and BPSA was determined by immunoassay. TABLE1 % [−2]pPSA % BPSA Lane #, Figure 1 ([−2]pPSA/Free PSA) (BPSA/Free PSA)2 (cancer) 95  7 3 (cancer) 42 12 4 (cancer) 49 16 5 (cancer) 40  5 6(cancer) 25 14 7 (biopsy-negative) 19 15 8 (biopsy-negative)  6 13 9(biopsy-negative)  8 25

[0125]FIG. 9 shows 2 different examples of how the ratio of[−2]pPSA/BPSA could be used to detect prostate cancer. In each examplethe biopsy-positive (i.e., cancer) and biopsy-negative samples containedessentially the same amount of total PSA in the serum. In example A,both samples contained about 10 ng/ml total PSA. The cancer samplecontained high [−2]pPSA and low BPSA compared to the PSA-matchedbiopsy-negative sample. This results in a [−2]pPSA/BPSA ratio for thecancer serum that is 25-fold higher than the biopsy-negative serum.

[0126] The serum samples in example B contained about 6 ng/ml PSA. ThisPSA value is in the diagnostic “gray zone” of 4-10 ng/ml where it isvery difficult to distinguish prostate cancer from benign disease. Whileboth samples contain comparable levels of BPSA, the [−2]pPSA wassignificantly elevated in the cancer serum. In this case the[−2]pPSA/BPSA ratio of the cancer serum was 4-fold higher than thebiopsy-negative serum, and provides a clear correlation with prostatecancer.

EXAMPLE 7 [−2]pPSA Is a stable, Inactive Form of pPSA

[0127] We have previously shown that hK2 and trypsin can activate[−5/−7]pPSA to mature PSA (see the commonly owned co-pending patentapplication Ser. Nos. 09/302,965, and 08/846,408). In the current study,we tested the [−4] and [−2]pPSA forms in order to determine if theseisoforms had altered susceptibility to activation. hK2 was incubatedwith each of the pPSA forms and the proportion of each form wasmonitored by HIC-HPLC as shown in FIG. 3B.

[0128]FIG. 10 shows the relative rate of pPSA activation by hK2. The[5/−7]pPSA was activated most rapidly, while the [−4]pPSA was activatedmore slowly. Most significantly, [−2]pPSA was not activated by hK2. FIG.11 shows the chromatographic profile of pPSA forms after an extendedincubation of 40% hK2 with the individual isoform of pPSA. After 5 h ofincubation at 37° C., [−2]pPSA still showed no evidence of conversion tomature PSA (FIG. 11A). After incubation for 2 h, the majority of the[−4]pPSA was converted (FIG. 11B). By contrast, the [−5] and [−7]pPSAforms were converted to mature PSA in less than an hour (FIG. 11C). Itshould be noted that the ratio of the [−5] and [−7]pPSA speciescontained within the 12 min peak in panel C was unchanged throughout theactivation process, as determined by N-terminal sequencing, indicatingthat these two species were biochemically indistinguishable assubstrates for hK2. Therefore, the major shift in HIC-HPLC retentiontime and in the activation kinetics of pPSA forms occurred upon removalof the single additional [−5] leucine residue. These differences betweenthe [−5] and the [−4]pPSA forms suggests that the [−5] leucine plays animportant role in the folding of pPSA.

[0129] In addition to hK2, we have previously shown that 1% trypsinrapidly activates pPSA in 15 min. We therefore tested trypsin for itsability to activate [−2]pPSA, since trypsin is a far more activeprotease than hK2, has a strong specificity for arginine (and lysine)residues, and is little affected in its hydrolytic activity by aminoacids adjacent to the P1 cleavage site, other than proline. After anextended incubation with trypsin, other internal arginine/lysine siteswere cleaved (as determined by N-terminal sequencing), but there waslittle or no cleavage to release the SR dipeptide of the [−2]pPSA (datanot shown).

[0130] Thus, the pro leader dipeptide on [−2]pPSA appears to beresistant to cleavage by proteases that are otherwise capable ofcleaving the [−4] and [−5/−7] pro leader peptides. In an experimentsimilar to that shown in FIG. 5, none of the pPSA isoforms formed acomplex with a 4× excess of ACT after 5 h of incubation at 37° C. (datanot shown). This demonstrated that all truncated and fill-length pPSAforms were enzymatically inactive and would thus be expected to remainas free PSA in serum.

EXAMPLE 8 Monoclonal Antibodies with Preferential Reactivity to ProPSA

[0131] Using the methods described, the following antibodies weregenerated in PS1Z fusion: PS1Z134, PS1Z120, PS1Z125 and PS1Z81. Table 1shows the results of screening at initial testing (proPSA alone), retestand expansion phases (comparison of proPSA response to PSA response).These monoclonal antibodies demonstrated elevated response to proPSA(approximately 2 fold) over response to PSA. This ability todifferentiate proPSA from PSA was maintained when the clones were grownin large size culture condition. TABLE 2 Specificity analysis of proPSAreactive hybridomas. Assay Response (A490) to proPSA vs PSA CellExpansion Initial screening Retest proPSA to PSA Clone ProPSA proPSA toPSA ratio ratio PS1Z134 + 2.3 2.0 PS1Z120 + 2.0 1.49 PS1Z125 + 2.2 1.58PS1Z81 + 2.0 1.35

EXAMPLE 9 Characterization of pPSA Monoclonal Antibodies Specific forpPSA

[0132] Monoclonal antibodies (mAbs) were prepared against full length[−7]pPSA by immunization with the chimeric recombinant proteinconsisting of hK2 with the APLILSR pro PSA leader peptide. Since thefirst 17 N-terminal amino acids of hK2 are identical to PSA, thesubstitution of the pPSA pro leader peptide onto hK2 provides advantagesin the immunization and screening procedures with pPSA since antibodiesto the remainder of the mature PSA protein are not induced. FIG. 12shows the sandwich immunoassay showing that 3 mAbs, PS2P206, PS2P309 andPS2P446, have a high specificity for pPSA compared to the mature form ofPSA.

[0133] In addition to the above, mAbs to the truncated forms of pPSA,specifically [−2]pPSA and [−4]pPSA were obtained by immunization withpeptides consisting of SRIVGGWECEK, and ILSRIVGGWECEK, respectively.FIG. 13, the top and middle panels, shows the specificity of 2 mAbseach. Each blot was cut in half, indicated by the dotted line, andprobed with 2 different mAbs (A and B). In the top panel, the 2 [−2]pPSAmAbs, PS2X094, and PS2X373, are shown. The lanes designated—2 indicates[−2]pPSA; −4, [−4]pPSA; −7, [−7]pPSA; and 10′ indicates mature PSA. Eachlane was loaded with 100 μg of protein. The blot shows that each of the[−2]pPSA mAbs has high specificity for [−2]pPSA and negligiblereactivity with the [−4]pPSA, [−7]pPSA, and mature PSA. In otherexperiments, these mAbs showed a cross-reactivity of about 20% formature PSA when the gels were run under reducing conditions, and about5% when the gels were run under non-reducing conditions. The middlepanel shows identical blots probed with 2 [−4]pPSA mAbs, PS2V41 1, andPS2V476. These mAbs show only minimal cross-reactivity to [−2]pPSA andno cross-reactivity to [−7]pPSA or mature PSA. The bottom panel in FIG.13 shows an identical blot probed with 2 [−7]pPSA m-Abs, PS2P309, andPS2P446. These are two of the mAbs described in FIG. 12. This blot showsthat under denaturing conditions on a Western blot, these mAbs haveequivalent reactivities with the [−4] and [−7]pPSA forms, but noreactivity to [−2]pPSA and mature PSA.

[0134] The three mAbs with selective pPSA specificities, PS2P446([−7]/[−4]pPSA specificity), PS2V476 ([−4]pPSA specificity), and PS2X373([−2]pPSA specificity), were also tested on their relative reactivitiesto each other, and to mature PSA in a sandwich assay, microtiter plate.In this case, the biotinylated Fab′ of mAb PS2J163 which binds to allPSA forms ²⁴ was bound to a streptavidin-coated microtiter plate, andincubated with the indicated form of pPSA as seen in FIG. 14. The above3 mAbs were then serially diluted and incubated with the different boundPSA and pPSA antigens. The binding of the pPSA mAbs was detected withanti-mouse-HRP antibodies. FIG. 14 shows that each of the pPSA mabs hadspecificity for their indicated form of pPSA. There is somecross-reactivity to other pPSA forms in some cases, but all shownegligible reactivity to mature PSA. The important result of thisexperiment is that it shows that mAbs raised to both pPSA proteins andpeptides are capable of reacting with good specificity to theappropriate native pPSA under conditions suitable for the development ofan immunoassay.

Discussion

[0135] The present invention has employed several novel approaches thathave resulted in the development of mAbs to detect and discriminatebetween 3 different isoforms of pPSA (FIGS. 12-14). These mAbs containedthe required properties of high sensitivity and specificity to thedifferent pPSA forms compared to mature PSA.

[0136] Using these mAbs, we were able to determine that different pPSAforms exist in serum, and that the [−2] appears to be the most prevalentform (FIG. 6). Just as importantly, it was found that [−2]pPSA compriseda significant percentage of the free PSA in prostate cancer serum,ranging from 25-95% of the free PSA (Table 1). Of equal importance, thisoccurred in the serum of men with total PSA values from 6-24 ng/ml.Thus, [−2]pPSA represents a significant and relevant isoform of PSA forthe study of prostate cancer. It is in the range below 10 ng/ml that itis most difficult to discriminate PCa from benign conditions such as BPHsince both conditions release PSA into the serum^(13,25). Repeatbiopsies are often performed in biopsy-negative men with elevated PSAlevels. This is because a positive biopsy is conclusive proof ofprostate cancer, while a negative biopsy may simply have missed theareas of cancer²⁶. For this reason the measurement of pPSA forms mayoffer additional diagnostic value.

[0137] In our study using pooled cancer serum containing 63 ng/ml totalPSA, we identified the truncated [−4]pPSA using HIC-HPLC (FIG. 4). In amore detailed study using pPSA mAbs and individual patient serum we haveshown that [−2]pPSA may be the more predominant form (FIG. 6). There is,however, no conflict with these results since, in FIG. 4, we usedchromatographic methodologies to identify the different forms of PSA.The HIC-HPLC approach required that elution fractions be collected andthe PSA measured by immunoassay. Because PSA and [−2]pPSA elute closely(see FIG. 3B), it was not possible to distinguish these 2 isoforms ofPSA in the FIG. 4 due to the loss of peak resolution incurred byfraction collection. Therefore, only the [−4]pPSA form was clearlyresolved from the PSA peak. It is not known, but it is likely, that thePSA peak in FIG. 4B contained some percentage of [−2]pPSA.

[0138] The reason for the enrichment of a stable, truncated form of pPSAin tissues and, ultimately, serum was suggested by the activationstudies. It has been previously demonstrated that pPSA can be activatedby hK2 and trypsin^(2,27,28) Since hK2 and PSA are co-localized in theprostate columnar epithelial cells²⁹, it has been speculated that hK2may be the endogenous protein responsible for the activation of PSA. Theprocess of pPSA activation is normally an extremely efficient processsince pPSA forms are undetectable in seminal plasma (data not shown).FIG. 10 shows that hK2 has no ability to activate [−2]pPSA and hasreduced activity on [−4]pPSA compared to [−5/−7]pPSA. Even trypsin,which has a much wider substrate specificity range and activates pPSA atleast 10× more rapidly than hK2, was unable to activate [−2]pPSA (datanot shown). An extended incubation with trypsin resulted in cleavage atother internal sites in PSA, without significant cleavage of theserine-arginine pro leader peptide. While there is no direct evidencethat hK2 is responsible for the activation of pPSA the failure of bothhK2 and trypsin to activate [−2]pPSA makes it less likely that [−2]pPSAremains a viable substrate for other activation proteases.

[0139] The present invention is the first description of the relativelevels of different pPSA forms in serum, and is in direct contrast toother reports in the literature that were unable to detect pPSA by othertechniques^(9,10), or were unable to develop the mAbs necessary toattempt pPSA detection in serum¹².

[0140] While not wanting to be bound by the theory, it is possible thatthe truncation of pPSA to [−2]pPSA is the result of post-translationalproteolytic cleavage. In our study of tissue extracts, we found that[−2]pPSA was the most elevated in cancer³⁰. In the transition zone, thesite of BPH, the median value of [−2]pPSA dropped to 0. This isimportant, since PSA leaks into the serum as the result of cancer andBPH. Since [−2]pPSA was not present in BPH transition zone tissue, itfollows that [−2]pPSA in the serum came from cancer tissues orsurrounding peripheral zone tissue. The immunostaining of prostatetissues (FIG. 7) adds important insight to our tissue extract results byconfirming the strong association of pPSA forms with cancerousepithelium. The results in FIG. 6 support this hypothesis by showingstrongly elevated levels of [−2]pPSA in PCa serum. In contrast, PSAreleased from the transition zone due to BPH would be expected tocontain little or no [−2]pPSA. While 5/5 cancer sera containedsignificant levels of [−2]pPSA, 2/3 biopsy negative, i.e., potential BPHserum, contained only trace levels of [−2]pPSA.

[0141] Since elevated levels of free PSA have been shown to bettercorrelate with a benign disease^(14,25,31), it may seem counterintuitive that cancer serum contains elevated levels of pPSA forms,which are also found as free PSA. However, FIG. 6 shows that pPSArepresents a minor percentage of the free PSA in benign disease and amajor percentage in cancer. The cancer samples had a lower % free PSAthan the benign samples, but a much higher relative percentage of thefree PSA was [−2]pPSA. As the % free PSA increased in thebiopsy-negative samples, it was apparently comprised of increasingamounts of inactive PSA forms other than [−2]pPSA. Thus, pPSA makes up aprogressively smaller percentage of the % free PSA that derives from abenign disease. Since men with prostate cancer can also develop BPH andvice versa, it will be important to determine the relative contributionof pPSA in each disease state.

[0142] [−2]pPSA, or other pPSA forms, could offer the greatestdiagnostic value as the percentage of total PSA, as a percentage of thefree PSA, or as an independent indicator where pPSA levels above acertain threshold have a high statistical correlation with cancer. Inthe latter case, it is conceivable that the presence of pPSA could offerdiagnostic value in serum containing any measurable level of pPSA, andespecially in serum with less than 4 ng/ml total PSA.

[0143] We have recently developed an immunoassay to detect serum BPSA,the BPH associated form of PSA. A pPSA immunoassay in combination withan assay for BPSA may add even greater discrimination of PCa from BPH.The results of the present invention show that proPSA, such as [−2]pPSA,is elevated in prostate cancer serum (FIG. 6) and that BPSA is elevatedin BPH serum (FIG. 8). In 2 examples of biopsy-positive andbiopsy-negative serum with matched total PSA, the ratio of [−2]pPSA/BPSAshowed a clear differential between cancer and benign disease (FIG. 9).

[0144] It is interesting to note that the [−2]pPSA/BPSA ratio showed asignificantly better differential between cancer and benign disease thanthe analysis of free and total PSA (an analysis currently used todistinguish prostate cancer from BPH). In FIG. 9, Example A, each samplecontained total PSA near 10 ng/ml, but very different percentages of %Free PSA. The cancer serum contained 9% Free PSA, while thebiopsy-negative serum contained 28% Free PSA. In numerous studies, %FreePSA values less than 10% are more highly correlated with cancer, whilevalues greater than 25% have a low correlation with cancer. For thesamples in Example A the ratio of [−2]pPSA/BPSA was 25-fold higher inthe cancer serum compared to the biopsy-negative serum.

[0145]FIG. 9, Example B shows serum samples that are more difficult tointerpret with the conventional free and total PSA assays. The total PSAis in the diagnostic “gray zone” of 4-10 ng/ml, and both samples alsocontained %Free PSA in the diagnostic %Free PSA “gray zone” between 10%and 25%. With current assay protocols employing only free and total PSAthere is no clear differential and no clear indication of prostatecancer in either sample. However, the [−2]pPSA was elevated in thecancer sample, and the [−2]pPSA/BPSA ratio was still 4-fold higher inthe cancer sample.

[0146] These examples indicate that the analysis of the [−2]pPSA andBPSA isoforms of PSA, and the ratio thereof, may add significantly tothe detection of prostate cancer.

[0147] The invention may be embodied in other specific forms withoutdeparting from its essential characteristics. The described embodimentsare to be considered in all respects only as illustrative and not asrestrictive. Indeed, those skilled in the art can readily envision andproduce further embodiments, based on the teachings herein, withoutundue experimentation. The scope of the invention is, therefore,indicated by the appended claims rather than by the foregoingdescription. All changes that come within the meaning and range of theequivalence of the claims are to be embraced within their scope.

[0148] References List:

[0149] 1. Catalona, W. J., Smith, D. S., Ratliff, T. L., Dodds, K. M.,Coplen, D. E., Yuan, J. J., Tetros, J. A., and Andriole, G. L.Measurement of prostate-specific antigen in serum as a screening testfor prostate cancer. N Engl J Med, 324: 1156-1161, 1991.

[0150] 2. Oesterling, J. E. Prostate-specific antigen: a criticalassessment of the most useful tumor marker for adenocarcinoma of theprostate. J Urol, 145: 907-923, 1991.

[0151] 3. Labrie, F., Dupont, A., Suburu, R., Cusan, L., Tremblay, M.,Gomez, J. L., and Emond, J. Serum prostate specific antigen aspre-screening test for prostate cancer. J Urol, 147: 846-851, 1992.

[0152] 4. Lilja, H., Christensson, A., Dahlen, U., Matikainen, M. T.,Nilsson, O., Pettersson, K., and Lovgren, T. Prostate-Specific Antigenin Serum Occurs Predominantly in Complex with α₁-antichymotrypsin. ClinChem, 37: 1618-1625, 1991.

[0153] 5. Stenman, U. H., Leinonen, J., Alfthan, H., Rannikko, S.,Tuhkanen, K., and Alfthan, O. A complex between prostate specificantigen and α₁-antichymotrypsin is the major form of prostate-specificantigen in serum of patients with prostatic cancer: assay of the compleximproves clinical sensitivity for cancer. Cancer Res., 51. 222-226,1991.

[0154] 6. Catalona, W. J., Partin, A. W., Slawin, K. M., Brawer, M. K.,Flanigan, R. C., Patel, A., Richie, J. P., deKemion, J. B., Walsh, P.C., Scardino, P. T., Lange, P. H., Subong, E. N., Parson, R. E., Gasior,G. H., Loveland, K. G., and Southwick, P. C. Use of the percentage offree prostate-specific antigen to enhance differentiation of prostatecancer from benign prostatic disease: a prospective multicenter clinicaltrial. Jama, 279: 1542-1547, 1998.

[0155] 7. Woodrum, D. L., Brawer, M. K., Partin, A. W., Catalona, W. J.,and Southwick, P. C. Interpretation of free prostate specific antigenclinical research studies for the detection of prostate cancer. J Urol,159: 5-12, 1998.

[0156] 8. Peter, J., Unverzagt, C., and Hoesel, W. Analysis of freeprostate-specific antigen (PSA) after chemical release from the complexwith alpha(1)-antichymotrypsin (PSA-ACT). Clin.Chem., 46: 474-482, 2000.

[0157] 9. Noldus, J., Chen, Z., and Stamey, T. Isolation andcharacterization of free form prostate specific antigen (f-PSA) in seraof men with prostate cancer. J Urol., 158: 1606-1609, 1997.

[0158] 10. Hilz, H., Noldus, J., Hammerer, P., Buck, F., Luck, M., andHuland, H. Molecular heterogeneity of free PSA in sera of patients withbenign and malignant prostate tumors [In Process Citation]. Eur Urol,36: 286-292, 1999.

[0159] 11. Paus, E., Nustad, K., and Bormer, O. P. Epitope mapping andaffinity estimation of 83 antibodies against prostate-specific antigen.Tumour.Biol., 20 Suppl 1: 52-69, 1999.

[0160] 12. Nurmikko, P., Vaisanen, V., Puironen, T., Lindgren, S.,Lilja, H., and Pettersson, K. Production and characterization of novelanti-prostate-specific antigen (PSA) monoclonal antibodies that Do notdetect internally cleaved Lysl45-Lys146 inactive PSA [In ProcessCitation]. Clin.Chem., 46: 1610-1618, 2000.

[0161] 13. Catalona, W. J., Smith, D. S., Ratliff, T. L., Dodds, K. M.,Coplen, D. E., Yuan, J. J., Tetros, J. A., and Andriole, G. L.Measurement of prostate-specific antigen in serum as a screening testfor prostate cancer. N Engl J Med, 324: 1156-1161, 1991.

[0162] 14. Woodrum, D. L., Brawer, M. K., Partin, A. W., Catalona, W.J., and Southwick, P. C. Interpretation of free prostate specificantigen clinical research studies for the detection of prostate cancer.J Urol, 159: 5-12, 1998.

[0163] 15. Berg, D. T., McClure, D. B., and Grinnell, B. W. Ela-responsive mammalian host/vector system for the stable high-levelexpression of secreted proteins. Nucleic Acids Res., 20: 5485-5486,1992.

[0164] 16. Wang, T. J., Linton, H. J., Sokoloff, R. L., Grauer, L. S.,Rittenhouse, H. G., and Wolfert, R. L. Antibody specificities for PSAand PSA fragments by SDS-PAGE Western blot analysis. Tumor Biology, 20:75-78, 1997.

[0165] 17. Finlay, J. A., Day, J. R., and Rittenhouse, H. G. Polyclonaland Monoclonal Antibodies to Prostate-Specific Antigen can Cross-Reactwith Human Kallikrein 2 and Human Kallikrein 1. Urology, 53: 746-751,1999.

[0166] 18. Kumar, A., Mikolajczyk, S. D., Goel, A. S., Millar, L. S.,and Saedi, M. S. Expression of pro form of Prostate-specific antigen bymammalian cells and its conversion to mature, active form by humankallikrein 2. Cancer Res, 57: 3111-3114, 1997.

[0167] 19. Christensson, A., Laurell, C. B., and Lilja, H. Enzymaticactivity of prostate-specific antigen and its reactions withextracellular serine proteinase inhibitors. Eur J Biochem, 194: 755-763,1990.

[0168] 20. Knott, C. L., Kuus-Reichel, K., Liu, R. S., and Wolfert, R.L. Development of antibodies for diagnostic assays. In: Principles andPractice of Immunoassay. Price, C. P. and Newman, D. J. 37-64.1997. NewYork, N.Y., Stockton Press. Ref Type: Serial (Book,Monograph)

[0169] 21. Kumar, A., Goel, A., Hill, T., Mikolajczyk, S., Millar, L.,Kuus-Reichel, K., and Saedi, M. Expression of human glandularkallikrein, hK2, in mammalian cells. Cancer Res., 56: 5397-5402, 1996.

[0170] 22. Kumar, A., Mikolajczyk, S. D., Goel, A. S., Millar, L. S.,and Saedi, M. S. Expression of pro form of Prostate-specific antigen bymammalian cells and its conversion to mature, active form by humankallikrein 2. Cancer Res, 57: 3111-3114, 1997.

[0171] 23. Mikolajczyk, S. D., Millar, L. S., Wang, T. J., Rittenhouse,H. G., Wolfert, R. L., Marks, L. S., Song, W., Wheeler, T. M., andSlawin, K. M. “BPSA,” a specific molecular form of freeprostate-specific antigen, is found predominantly in the transition zoneof patients with nodular benign prostatic hyperplasia. Urology, 55:41-45, 2000.

[0172] 24. Wang, T. J., Slawin, K. M., Rittenhouse, H. G., Millar, L.S., and Mikolajczyk, S. D. Benign prostatic hyperplasia-associatedprostate-specific antigen (BPSA) shows unique immunoreactivity withanti-PSA monoclonal antibodies. Eur.J.Biochem., 267: 4040-4045, 2000.

[0173] 25. Catalona, W. J., Partin, A. W., Slawin, K. M., Brawer, M. K.,Flanigan, R. C., Patel, A., Richie, J. P., deKemion, J. B., Walsh, P.C., Scardino, P. T., Lange, P. H., Subong, E. N., Parson, R. E., Gasior,G. H., Loveland, K. G., and Southwick, P. C. Use of the percentage offree prostate-specific antigen to enhance differentiation of prostatecancer from benign prostatic disease: a prospective multicenter clinicaltrial. Jama, 279: 1542-1547, 1998.

[0174] 26. Naughton, C. K., Smith, D. S., Humphrey, P. A., Catalona, W.J., and Keetch, D. W. Clinical and pathologic tumor characteristics ofprostate cancer as a function of the number of biopsy cores: aretrospective study. Urology, 52: 808-813, 1998.

[0175] 27. Lovgren, J., Rajakoski, K., Karp, M., Lundwall, A., andLilja, H. Activation of the zymogen form of prostate-specific antigen byhuman glandular kallikrein 2. Biochem.Biophys.Res.Comm., 238: 549-555,1997.

[0176] 28. Takayama, T. K., Fujikawa, K., and Davie, E. W.Characterization of the precursor of prostate-specific antigen-activation by trypsin and by human glandular kallikrein. J.Biol.Chem.,272: 21582-21588, 1997.

[0177] 29. Darson, M. F., Parcelli, A., Roche, P., Rittenhouse, H. G.,Wolfert, R. L., Young, C. Y. F., Klee, G. G., Tindall, D. J., andBostwick, D. G. Human Glandular Kallikrein 2 (hK2) expression inprostatic intraepithelial neoplasia and adenocarcinoma: a novel prostatecancer marker. Urology, 49(6): 857-862, 1997.

[0178] 30. Mikolajczyk, S. D., Millar, L. S., Wang, T. J., Rittenhouse,H. G., Marks, L. S., Song, W., Wheeler, T. M., and Slawin, K. M. Aprecursor form of prostate-specific antigen is more highly elevated inprostate cancer compared with benign transition zone prostate tissue.Cancer Res, 60: 756-759, 2000.

[0179] 31. Catalona, W. J. Clinical utility of measurements of free andtotal prostate-specific antigen (PSA) : a review. Prostate, Supplemnent7: 64-69, 1996.

[0180] 32. Linnet K. Necessary sample size for method comparison studiesbased on regression analysis. Clin Chem 1999;45:882-94.

[0181] 33. Linnet K. Evaluation of regression procedures for methodscomparison studies. Clin Chem 1993;39:424-32.

[0182] 34. Bland J M, Altman DG. Statistical methods for assessingagreement between two methods of clinical measurement. Lancet1986;i:307-10.

[0183] 35. Reid M C, Lachs M S, Feinstein A R. Use of methodologicstandards in diagnostic test research. Getting better but still notgood. JAMA 1995;274:645-51.

[0184] 36. Krouwer J S. Cumulative distribution analysis graphs—analternative to ROC curves [Tech Brief]. Clin Chem 1987;33:2305-6.

[0185] 37. Albert A. On the use and computation of likelihood ratios inclinical chemistry. Clin Chem 1982;28:1113-9.

[0186] 38. Solberg H E. Discriminant analysis. Crit Rev Clin Lab Sci1978;9:209-42.

[0187] 39. Matthews J N S, Altman D G, Campbell M J, Royston P. Analysisof serial measurements in medical research. Br Med J 1990;300:230-5.

We claim:
 1. An antibody that specifically binds to a pPSA.
 2. Theantibody of claim 1, wherein the antibody specifically binds to a pPSAselected from a group consisting of [−2]pPSA, [−4]pPSA, and [−7]pPSA. 3.The antibody of claim 2, wherein the antibody specifically binds to[−2]pPSA.
 4. The antibody of claim 1 which is a monoclonal antibody. 5.The antibody of claim 4 selected from a group consisting of PS2P206,PS2P309, PS2P446, PS2P031, PS2P401, PS2P167, PS2P125, PS2P134, PS2X094,PS2X373, PS2X199, PS2X458, PS2X572, PS2V41 1, and PS2V476.
 6. Ahybridoma cell line producing an antibody that specifically binds topPSA.
 7. The hybridoma cell line of claim 6 which produces an antibodyspecifically binds to a pPSA selected from a group consisting of[−2]pPSA, [−4]pPSA. and [−7]pPSA.
 8. The hybridoma cell line of claim 6producing a monoclonal antibody selected from a group consisting of ofPS2P206, PS2P309, PS2P446, PS2P031, PS2P401, PS2P167, PS2P125, PS2P134,PS2X094, PS2X373, PS2X199, PS2X458 PS2X572, PS2V411, and PS2V476.
 9. Adiagnostic kit for detecting or determining pPSA in a sample comprisinga known amount of an antibody that specifically binds to pPSA.
 10. Thediagnostic kit of claim 9, wherein said antibody is detectably labeledor capable of being labeled.
 11. The diagnostic kit of claim 9, whereinsaid antibody is attached to a solid support or capable of beingattached to a solid support.
 12. The diagnostic kit of claim 10 furthercomprising a second antibody that is attached to a solid support orcapable of being attached to a solid support.
 13. The diagnostic kit ofclaim 11 further comprising a second antibody that is detectably labeledor capable of being labeled.
 14. The diagnostic kit of claim 9, whereinthe sample is a sample of human physiological fluid.
 15. The diagnostickit of claim 10, wherein the physiological fluid is selected from agroup consisting of blood, serum, seminal plasma, urine or plasma. 16.The diagnostic kit of claim 9 further comprising a solid phase capableof having said antibody attached thereto.
 17. The diagnostic kit ofclaim 9, wherein the sample is a mammalian tissue sample.
 18. Thediagnostic kit of claim 9, wherein the antibody is a monoclonal antibodyselected from a group consisting of PS2P206, PS2P309, PS2P446, PS2P031,PS2P401, PS2P167, PS2P125, PS2P134, PS2X094, PS2X373, PS2X199, PS2X458,PS2X572, PS2V41 1, and PS2V476.
 19. The diagnostic kit of claim 9,wherein the antibody is specific for [−2]pPSA.
 20. The diagnostic kit ofclaim 15, wherein the antibody is PSX373.
 21. The diagnostic kit ofclaim 9, wherein the antibody is specific for [−4]pPSA.
 22. Thediagnostic kit of claim 17, wherein the antibody is PS2V476.
 23. Thediagnostic kit of claim 9, wherein the antibody is specific for[−7]pPSA.
 24. The diagnostic kit of claim 19, wherein the antibody isPS2P446.